The estimation of emission lifetimes (excited state lifetimes), e. g. based on fluorescence or phosphorescence emission is an essential technique in basic and applied science. Lifetime (tau, τ) determinations provide sensitive measures of binding and other molecular interactions and conformational states of macromolecules, such as proteins and nucleic acids, as well as of physical properties of their microenvironment (e.g. viscosity, polarity, pH). A prominent application of time-resolved spectroscopy is FRET (Förster Resonance Energy Transfer), in which the quantum yield (and thus lifetime) of a donor fluorophore changes according to the orientation and distance (6th power dependence) of a donor molecule to an acceptor molecule.
For measuring emission lifetimes, the sample under investigation is illuminated with pulse-shaped excitation light. An emission response from the sample is detected using a detector device with time-resolution. The detector device creates a temporal detector response function (output signal function), e. g. representing the time dependency of the emission response from the sample, on the basis of which the lifetime(s) of the electronically excited states in the sample is/are calculated.
Emission lifetimes can be measured at a single sample location or with spatial resolution at a plurality of sample locations. In the latter case, an imaging modality is provided which in the case of fluorescence is called Fluorescence Lifetime Imaging (FLIM). FLIM is applied extensively in biological imaging (microscopy) in order to determine the conformation, activation, interactions and redistributions of key molecules involved in signal transduction. In biotechnology, fluorescence decay measurements are used for high-throughput screening (e.g. via binding assays) of prospective diagnostic or therapeutic molecules with designated targets.
The decay of the electronic (singlet or triplet) excited state is generally characterized by a defined probability given by the combination of various depopulation processes (in particular spontaneous emission, resonance energy transfer, non-radiative decay) and is generally exponential in nature after the cessation of the excitation source. The conventional methods for lifetime determination seek to extract the exponential time constant (reciprocal of the lifetime τ), or multiple time constants in the case of heterogeneous systems, from the time course of the emissive decay curves, which are included in the measured detector response functions.
The available techniques are divided into those appropriate for discrete samples (e.g. cuvettes, microtiter plate wells) or complex specimens under the microscope. The latter case constitutes the imaging modality FLIM and requires the use of either a scanning system with one (or a few) detectors or a camera capable of acquiring a temporal sequence of 2D images. The extensive array of FLIM technologies and their comparative merits and biological applications are summarized by a large number of reviews, e.g. by W. Becker in “J. Microsc.” 247: 119-136 (2012) or M. Y. Berezin et al. in “Chem. Rev.” 110:2641-2684 (2010. There exist numerous related implementations of FLIM in the material sciences and other applied fields.
Scanning systems incorporated into confocal microscopes generally utilize the time-correlated single-photon counting (TCSPC) technique, which provides great inherent sensitivity and temporal resolution. Camera-based systems operating in the time domain and using a train of very narrow, excitation pulses require a gated-intensifier front-end defining programmable temporal relationships between a detection window and an excitation pulse. Similar instruments operating in the frequency domain employ periodic (sinusoidal) excitation and phase-sensitive detection. In this case, the lifetimes are derived from the modulation amplitudes and phases of the detected signals relative to that of the excitation source. Newer emerging camera systems perform phase-sensitive detection directly on the detector chip surface.
The experimental difficulty in conventional time-domain measurements, in particular FLIM, featuring excitation light pulses of narrow but finite width results from the fact that the detector response function is given by the convolution of the excitation light pulse with the exponential emissive decay function, and is thus arbitrarily complex in shape. As a consequence, short excitation light pulses (duration less to or comparable to the decay time) are required.
Furthermore, data analysis of the detector response function for deriving the decay time requires deconvolution of the response and/or restriction of the data set to a final segment of the decay. In both cases, complex mathematical procedures based on iterative minimization are involved. These require considerable computation time, even with current computers, extending to minutes in the event that more than a single decay component is present. Most FLIM applications are based on the mean decay lifetime computed from the individual decay times and their corresponding amplitudes. Deriving a mean value still requires a complete decay analysis in order to obtain the number of components and their parameters. This limitation applies to all current FLIM or single channel lifetime techniques.